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The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators.Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain,which serves as a promising drug target for cancers and immune system disorders.Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported,but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking.Here,we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide.Through an HTS assay against an in-house chemical library containing 20 000 compounds,compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM.This compound bound to CBP BrD with a KD value of 4.01 μM in the surface plasmon resonance assay.Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168.At the celluslar level,DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 μM and markedly downregulated the expression of the c-Myc in the cells.Taken together,the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research.In addition,this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.